Antifungal activity of Terminalia chebula fruit extracts

The present study was aimed to investigate the anticandidal and antifungal potential of dried fruit extracts of Terminalia chebula against Candida albicans, C. tropicalis C. glabrata, C. krusei, C. parapsilosis, and Aspergillus flavus, A. niger, A. fumigatus, Trichophyton mentagrophytes, T. rubrum, Microsporum gypseum. Phytochemical analysis of methanol extracts of T. chebula dried fruits showed the presence of flavonoids, alkaloids, glycosides, saponins, tannins, terpenoids and steroids. Among the tested four extracts, the methanol extracts of T. chebula dried fruits exhibited the highest antifungal activity and their inhibition zone was ranged between 7.5 to 19.5mm. MIC and MFC values were between 62.5-250μg/ml and 250-500μg/ml respectively. Zone of inhibition (19.5 mm), MIC (62.5μg/ml) and MFC (125μg/ml) values observed in methanolic extracts of T. chebula dried fruits against A. fumigates and T. mentagrophytes. Our findings proved that methanolic extracts of T. chebula dried fruits were possessed substantial anticandidal and antifungal properties.


INTRODUCTION
In tribal community, dermal and mucous infections are the most common type of infections occurs due their poor personal hygiene including food habits, sanitation and lacking of clean drinking water. The most common dermal fungal pathogens are belongs to dermatophytes and Candida sp. [1,2]. Among the recent fungal infection case studies, >90% infections are due to Candida albicans, which leads to Candidiasis [3]. Due to the affiliations between domesticated animals and humans often end with either ringworm or tinea infections. Dermatophytic species generally grows on the outer surface such as nails, hair and skin on humans as well as domestic animals appendages also. Dermatophytes specifically infest keratin protein present in hair, hooves, beaks and skin. Dermatophyte microbes belong to three genuses such as Trichophyton, Microsporum and Epidermophyton [4].
Another pathogen, Aspergillus fumigates also cause deleterious fungal infections especially in less immunocompetent humans patients which eventually end with Aspergillosis [5,6]. Similarly, immunocompetent individuals are more susceptible towards Cryptococcus neoformans, Candida albicans, Asperillgus fumigatus and Histoplasma capsulatum [7]. Recently, long term antifungal therapies prone to microbial evolution and became a multi drug resistant strain against broad spectrum of antibiotics which currently used in drug therapies against fungal infection [8]. Indeed, conventional antifungal agents from medicinal plants were used as an effective alternative source.
Various human and plant microbes such as Corynebacterium accolans, C. albicans, Staphylococcus aureus and Erwinia carotovora were treated with polyphenolic ellagic acid, gallic acid and corilagin [9]. Terminalia chebula Retzius belongs to the Family Combretaceae which is native to India and Southeast Asia. It is commonly called as black myroblans [10]. This plant popularly used in folk medicine either alone or with other herbs and also used as cardiotonic, denrifrice, stomache tonic, laxative and purgative. T. chebula fruits are used to treat skin infections, diabetes, digestive disorders, burns, and kidney dysfunction and eye diseases [11,10]. Thus, the current study was undertaken to analyze the antifungal activity of various solvent extracts of T. chebula dried fruits.

Collection and Preparation of Crude Extracts
The experimental plant Terminalia chebula Retzius (Combretaceae) were collected from Kovilur Panchyath at Javadhu hills of Eastern Ghats ( Figure 1) and the fruits were utilized in this study. T. chebula fruits washed with tap water, 10% of Sodium hypochlorite solution and finally rinsed with distilled water. The dried fruits were grounded finely into powder. In Soxhlet apparatus (>78°C), 500gms of fine powder was mixed with non-polar to polar solvents viz., hexane, ethyl acetate, chloroform and methanol for 72hours of each solvent. In a rotary evaporator, (Heidolph, Germany) the solvents were evaporated under vacuum and the dried extracts were stored (4 °C).

Antifungal Assays
Disc diffusion method T. chebula dried fruits crude extracts antifungal properties were studied by disc diffusion method [12]. For susceptibility test, 20ml SDA medium poured into Petri plates for yeast and filamentous fungi. 100μl of fungal suspension containing 10 6 CFU/ml and 10 4 spore/mL for yeast and mould fungi respectively were inoculated into the media. Under aseptic conditions, sterile (HiMedia) paper disc (6mm) were impregnated with 20μl of disparate extract concentrations (1000, 500 and 250μg/disc) dissolved in 10% DMSO and placed on the agar plates.
For positive control, Amphotericin -B (100units/disc) and Ketoconazole (10μg/disc) for yeast and Aspergillus, dermatophytes were used and 10% DMSO used as negative control. Finally, the inoculated plates were incubated for 24 -48h at 28 °C for yeast, for 72-96 h for Aspergillus sp. and 4-7 days with dermatophytes at 30°C. Inhibition zone was measured in mms.

Determination of Minimum Inhibitory Concentration (MIC)
T. chebula dried fruit extracts MIC was determined by broth micro dilution technique as recommended by CLSI M27-A3 [13] and M38-A2 [14] for yeasts and filamentous fungi, respectively. The MIC values were analyzed in RPMI-1640 (pH 7.0, Himedia, Mumbai) which is composed of L-glutamine with morpholine propane sulfonic acid (MOPS) without NaHCO 3 . 50mg/ml (20μL stock) of crude extracts mixed with 10% DMSO and dissolved in 980μl of RPMI-1640 medium solution. From this, solutions were two fold serial diluted in the ranges from 500 to 15.7μg/mL. In 96 well microtitre plates, 200μL of solution was poured into first well and then 100μL transferred to the next well containing 100μL of RPMI-1640, and the serially diluted to the other wells.
100μL of inoculum suspension was transmitted to each well to achieve a concentration of approximately 0.5-2.5×10 3 CFU/mL and 0.4-5×10 4 CFU/mL for yeasts and filamentous fungi. Without inoculum only sterile water was added for control well. The microtitre plates were incubated for 24-48 hrs at 28 °C for yeast and for 72-96 hrs for Aspergillus sp. and 4-7 days with dermatophytes at 30°C. Growth inhibition of different concentrations of crude extracts on Candida, Aspergillus and dermatophytic strains were compared with control results and the lowest concentration was recorded as MIC.

Determination of Minimum Fungicidal Concentration (MFC)
From each MIC titre well, SDA plates were inoculated with a loop of samples and then incubated for 24-48hrs at 28°C for yeast and for 72-96hrs for Aspergillus and 4-7 days for dermatophytes at 30°C. The lowest concentration of the extract which inhibits visible fungal growth after incubation was recorded as MFC.

Statistical Analysis
A result of statistical analysis was performed by using SPSS software 16.0 version (SPSS Inc., Chicago, IL, USA). The antifungal activity between the crude extracts and their significance were studied by student's t-test and their mean comparison was performed by one-way analysis of variance (ANOVA) and Duncan post hoc test. P value <0.05 was considered as statistically significant and the results were expressed as mean ± SD.

RESULTS
Hexane, ethyl acetate, chloroform and methanol extracts of T. chebula dried fruits were used to study the phytochemicals such as flavonoids, saponins, alkaloids, glycosides, terpenoids, steroids and tannins. Among the various extracts, methanolic extract of T. chebula dried fruits showed the strong presence of phytochemicals. Ethyl acetate extract contains all the phytochemicals tested except glycosides and steroids. Whereas, the chloroform extract contains only alkaloids, flavonoids, glycosides and tannins other tested phytochemicals are absent. The presence of alkaloids, flavonoids and glycosides was observed in the hexane extract (Table 1).

DISCUSSION
Based on the presence of secondary metabolites, the medicinal plants were used to treat human ailments. Recently, traditional pharmacological and ethnomedical approaches were received great attention and recognition in modern medicine due to the prospective medicinal plants [15].
In this studies, antifungal activity of hexane, ethyl acetate, chloroform and methanol extracts of T. chebula dried fruit were tested. All the tested microbial strains showed varied degree of inhibitions. T. chebula dried fruit methanol extract exhibited good activity against T. mentagrophytes [Mean inhibition zone (19.5mm), MIC (62.5μg/ml) and MFC (125μg/ml)] followed by ethyl acetate, chloroform and hexane extracts.
Aqueous extracts of T. chebula showed high antifungal activities whereas MIC and MFC values were found as 3.125μg/ml and 12.5μg/ml respectively.
T. chebula leaf extracts possessed antibacterial activities against four Gram positive bacterial strains such as Enterococcus faedalis, Bacillus subtillis, Corynebacterium, Staphylococcus aureus and three Gram-negative bacterial strains such as Klebsiella pneumonia, Shigella boydii and Salmonella typhi [17]. The aqueous extract of T. chebula showed antifungal activity against various dermatophytes and yeast (Floccosum, Tricophyton rubrum, Microsporum gypseum, Epidermophyton and C. albicans) [18,19,20]. In the present investigation, the methanol extract of T. chebula fruit possessed antifungal activity against few Aspergillus species, Candida species and Dermatophytic strains than other solvent extracts like hexane, chloroform and ethyl acetate which was similar to the previous report [23]. The methanol extract of Gnaphalium polycaulon leaf exhibited high antifungal activity against Aspergillus flavus, A. fumigates, A. oryzae, Candida albicans and Penicilium notatum.
In this study, methanol estract of T. chebula dried fruits showed highest antifungal activity due to their alkaloids, glycosides, saponins, tannins, flavonoids, terpenoids and steroids. In plants, alkaloids, tannins, flavonoids and many aromatic compounds or secondary metabolites which is involved in defense mechanism against invading microorganisms and various predators like herbivores and insects [28]. Similar results reported on 600μg/ml of ripened T.chebula fruits extracts against T. mentagrophytes [29].
The results of the present study evident the importance of T. chebula dried fruit extracts against various fungal pathogens. Further study on the isolation antifungal molecule and it's characterization from the methanol extract of T. chebula dried fruits are under process.